Lucifer Yellow dyes

The affinity-purified connexin-32 was incorporated into unilamellar liposomes as before. The connexin-32 induced a sucrose permeability in liposomes, as assayed by the density-shift technique, and gave results essentially identical to those in Figure 2. Liposomes that were sucrose-permeable did not retain the dye Lucifer Yellow (retained by the sucrose-impermeable liposomes), which is near the upper size-permeability limit for gap junction channels (19, 108, 109). The fraction of the liposomes that were permeable to sucrose decreased by a factor of 4 when the pH in the gradients was changed from 7.5 to 6.0. This effect was partially reversible. 

In xanthenes, even if all one-photon allowed transitions are also two-photon allowed, the shape of the bands and their relative intensities are very different in the IPA and 2PA spectra [76,78]. This is not the case for other laser dyes and chromophores, for which the two spectra are almost identical (if represented as a function of the total transition energy), showing peaks in the same position and with very similar band shapes. Some example of chromophores in this category are coumarin 307 [78], coumarin 102 [80], 7-hydroxycoumarin [81], lucifer yellow [78], and cascade blue [78]. 

Structural formula for Lucifer dyes For Lucifer yellow R = —NH—CO—NH—NH2... 

Lucifer Yellow probes are water-soluble to at least 1.5%. The absorbance maximum of the derivatives occurs at about 426—428 nm with an emission peak at about 530—535 nm, in the yellow region of the spectrum. The quantum yield of Lucifer dyes is about 0.25. The good intensity of luminosity from these dyes makes possible detection of small quantities of labeled molecules intracellularly. The fluorescent conjugates are readily visible in living cells at concentrations that are nontoxic to cell viability. The low molecular weight and water solubility of these dyes allow passage of labeled compounds from one cell to another, potentially revealing molecular relationships... 

Fig. 4.4. Lucifer yellow carbohydrazide dye indicating water flow applied to a deep-root chamber, and detected in mycorrhizal hyphae in a hyphal chamber after crossing airgaps that restrict diffusion. Panel A is an AM hypha that transported the dye during the night (hydraulic lift). Panel B shows a mycorrhizal fungus hydrophilic tip, with hydraulically lifted water exuding out the tip onto a piece of organic detritus. Photographs by Louise Egerton-Warburton and details of the experiment can be found in Querejeta et al. (2003).

Usually, a 4-10% solution of Lucifer Yellow CH in lithium chloride is iontophoreticaUy injected through glass capillaries by passing current (1-3 nA) pulses (50-500 nsec) at 10 or 1 Hz, respectively (Miller and Goodenough, 1985 Schuetze and Good-enough, 1982). Transfer of the dye into the surrounding cells can be observed under a fluorescence microscope about 10 min after the injection. If required, cells can be fixed after dye injection by incubation for at least 20 min in 4% paraformaldehyde in PBS (Ren et al., 1990). 

Lil) of 0.05% of Lucifer Yellow CH and Rhodamine dextran (mw 10,000) in PBS at room temperature. The dish is swirled to distribute the solution evenly, and then cells in the culture are scraped off the dish with a cell scraper. Cells are immediately resuspended in complete culture medium, rinsed at least twice by centrifugation to remove unincorporated macromolecules, and replated on a new dish containing a culture of cells which are the potential recipients of the dye via gap junction formation. After 1 h of incubation at 37°C, the dish can be examined under an epifluorescence phase microscope at the appropriate wavelengths. 

A general synthesis of 3,6-disulphonated 4-aminonaphtl limides (108), the Lucifer Yellow dyes, is... 

Paternostro MA, Reyher CKH, Brunjes PC. 1995. Intracellular injections of Lucifer Yellow into lightly fixed mitral cells reveal neuronal dye-coupling in the developing rat olfactory bulb. Dev Brain Res 84 1-10. 

Lucifer yellow dye 4% (w/v) Lucifer yellow in aqueous solution. 

When a cell body of the appropriate size and position is made visible, impale it with a microelectrode and perform electrophysiological recordings to determine its identity (see Note 8). If desired, dye-mark physiologically identiried cells with Lucifer yellow to ensure that the correct cells are removed (see Note 9 and Fig. 1). 

The octopamine content per cell is calculated by subtracting the background level detected in a matched blank tube from the level detected in the sample. The resulting value is then divided by the number of cells in the sample The mean octopamine content for these four samples is 27.07 pg/cell (0 143 pmol/cell). Dye marked cells were labeled with Lucifer yellow ND not detectable... 

To confirm that the cell bodies analyzed were from PM4 neurons, Lucifer yellow (a fluorescent dye) was injected (using 5 nA hyperpolanzing pulses of 100 ms duration at 5 Hz for 5-10 min) into the cell bodies prior to their removal. After removal of the cell bodies, the brain was fixed for 1 h in 4% (w/v) paraformaldehyde in PBS before processing with anti-Lucifer yellow antiserum (Molecular Probes, Leiden, The Netherlands). In all such preparations, processes in the protocerebrum and optic lobe, with the characteristic anatomical features of PM4, were stained (Fig. 1). We find that the presence of Lucifer yellow m the cell body... 

In most experimental situations, a choice of fluorochromes is available. Where two fluorochromes are used in the same specimen, it is important that their fluorescence signals be clearly distinguished (for example, different populations of dye-labeled axons or different immunolabels). It is important to compare the specifications of the excitation and emission filters with the absorption and emission spectra of the fluorochromes chosen. Other factors that should be taken into account are the brightness and photostability of a particular fluorochrome. Some fluorescent dyes survive fixation better than others. For example, for intracellular dye injection, the highly fluorescent 5,6-carboxyfluorescein cannot be fixed, whereas the relatively less bright lucifer yellow can be cross-linked by paraformaldehyde fixation. 

Figure 5.15 Anionic dyes used in saccharide sensing. All compounds are shown as purchased or prepared. MPTS = methoxypyrenetrisulfonic acid, trisulfonate PTCA = perylenetetracarboxylic acid, tetracarboxylate HPTS = hydroxypyrenetrisulfonic acid, trisulfonate APTS = amino-pyrenetrisulfonic acid, trisulfonate fluorescein-SA = fluorescein-5-(and-6-)sulfonic acid lucifer yellow-I = lucifer yellow iodoacetamide SR-B = sulforhodamine-B HPTS(Lys)3 = hydroxypyrenetri(lysine) sulfonamide TCPP = tetrakis(4-carboxyphenyl)porphine TSPP = tetrakis(4-sulfophenyl)porphine.

Quantification of cell-to-cell communication. Flow cytometry meikes it easy to quantify cell-to-cell communication. As an example work of Kavanagh may be cited. Cells are scrape-loaded with Lucifer yellow with or without rhodamine labelled dextran. The transfer of the two dyes between donor and recipient cells can be studied easily using two colour fluorescence flow cytometry. 

Electrodes were made from two-barrel borosilicate glass tubing with filaments (WPI). One barrel was filled with 3 M KCl (2-10 MU) for stimulation and recording. For microejections, the other barrel was filled with 5% Lucifer Yellow (Lucifer Yellow CH, lithium salt. Molecular Probes) in 150 mM LiCl or with lOmM Alexa fluor 488 (hydrazide, sodium salt. Molecular Probes) in 200 mM KCl. When the double-barrel microelectrode was inserted into a cell, dye ejection was accomplished with 100-msec negative current pulses (1-50 nA) delivered through the dye-filled barrel and applied for 1-5 min at 100-msec intervals. After dye ejection the tissue was fixed. 

Figure 8 Left Diagram summarizing intercellular coupling between different structures in the carotid body. GC, glomus cells SC, sustentacular cells NT, carotid nerve terminal. Arrows show points (gap junctions) where there is transfer of information from one cell to another. Note that all structures are interconnected. Right A Lucifer Yellow microinjection into a cell with a complex shape stains this element and dye diffuses to many other structures in the organ. Clearly, some are glomus cells. The injected structure may be a sustentacular cell.

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